Peptide-targeted polyglutamic acid doxorubicin conjugates for the treatment of alpha(v)beta(6)-positive cancers

Abstract

Most chemotherapeutics exert their effects on tumor cells as well as their healthy counterparts, resulting in dose limiting side effects. Cell-specific delivery of therapeutics can increase the therapeutic window for treatment by maintaining the therapeutic efficacy while decreasing the untoward side effects. We have previously identified a peptide, named H2009.1, which binds to the integrin alpha(v)beta(6). Here, we report the synthesis of a peptide targeted polyglutamic acid polymer in which the high affinity alpha(v)beta(6)-specific tetrameric H2009.1 peptide is incorporated via a thioether at the N-terminus of a 15 amino acid polymer of glutamic acid. Doxorubicin is incorporated into the polymer via an acid-labile hydrazone bond. Payloads of four doxorubicin molecules per targeting agent are achieved. The drug is released at pH 4.0 and 5.6 but the conjugate is stable at pH 7.0. The conjugate is selectively internalized into alpha(v)beta(6) positive cells as witnessed by flow cytometric analysis and fluorescent microscopy. Cellular uptake is mediated by the H2009.1 peptide, as no internalization of the doxorubicin-PG polymer is observed when it is conjugated to a scrambled sequence control peptide. Importantly, the conjugate is more cytotoxic toward a targeted cell than a cell line that does not express the integrin.

Figure 1

pH dependent release of doxorubicin from the polyglutamic acid-H2009.1 peptide conjugate. Polyglutamic acid-H2009.1-doxorubicin conjugate was dissolved in aqueous buffer at pH 7.0, pH 5.6, or pH 4.0 and incubated at 37 °C. At the indicated times, aliquots were removed and the ratio of unconjugated to conjugated doxorubicin was assessed by reverse phase HPLC on an analytical C18 column.

Figure 2

Cell targeted delivery of doxorubicin mediated by a cell-specific peptide. H2009 (panel A) and H1299 (panel B) cells were incubated for 2 h in the presence of 30 µM unconjugated doxorubicin (red line) or doxorubicin linked to the poly glutamic acid-H2009.1 (blue line) peptide conjugate or scrambled peptide conjugate (black line). At the end of this incubation period, the drug was removed; cells were washed with PBS⁺/0.1% BSA 4 times as well as twice with 20 mM HCl-glycine, pH 2.2. Uptake of doxorubicin into cells was assessed by flow cytometry, counting 10 000 events per sample. Results obtained with untreated cells are shown in gray shaded area.

Figure 3

Cell targeted delivery of doxorubicin preferentially kills α_Vβ₆-integrin positive tumor cells. H1299 (■ - black, red) and H2009 (● - black, red) cells were incubated in the presence of varying concentrations (200 nM to 5 µM) unconjugated doxorubicin (black lines) or doxorubicin linked to the polyglutamic acid-H2009.1 peptide conjugate (red lines). After 24 h, the drug was removed; cells were washed and incubated in fresh complete media for 72 h. Cell viability was determined using the Cell Titer-Glo assay (Promega Corp). The results shown are an average of 3 independent experiments. Pairwise comparison of cell viability after free dox treatment of both cell types did not demonstrate any statistical difference whereas the difference in the viability of these two cell types after poly-glu-dox-H2009.1 conjugate treatment was statistically significant (p = 0.033).

Figure 4

Cell targeted delivery of doxorubicin mediated by a cell-specific peptide. H1299 (A and B) and H2009 (C and D) cells were incubated in the presence of 5 µM unconjugated doxorubicin (A and C) or doxorubicin linked to the polyglutamic acid-H2009.1 peptide conjugate (B and D). After 24 h, the drug was removed; cells were washed and incubated in fresh complete media for 2 h. Cells were observed by phase contrast and fluorescence microscopy at 400× magnification. Overlays of the resulting images are shown.

Scheme 1. Synthesis of Targeted PG-Doxorubicin Conjugatea

^a The structure of the tetrameric H2009.1 peptide is shown in the inset.